Multicellular organisms have evolved elaborate mechanisms to enable differential and cell-type specific expression of genes. Epigenetics refers to these heritable changes in how DNA is accessed in different cell-types and during development and differentiation. The template upon which the epigenome is written is chromatin - the complex of histone proteins, RNA and DNA that efficiently package the genome in an appropriately accessible state within each cell. The state of chromatin, and therefore access to the genetic code, is largely regulated by specific chemical modifications to histone proteins and DNA, and the recognition of these marks by other proteins and protein complexes. Our understanding of chromatin function is in its infancy and chemical biology can play a central role in advancing scientific knowledge and assessing therapeutic opportunities. Specifically, cell penetrant, high-quality chemical probes that modulate the regulation of chromatin state are of great significance in the fields of epigenetics, oncology, developmental biology, neurology, stem cell fate and regenerative medicine. The recognition of the methylation-state of lysine residues in histones is a critical event in chromatin regulation. For example, different lysine methylation marks (KMe) are associated with active (histone 3, lysine 4 dimethylation, i.e. - H3K4Me2) and repressed transcriptional states (H3K9Me2). The more than 200 methyl-lysine binding proteins in the human genome represent a relatively unexplored set of targets for intervention with small molecules. The overarching objectives of this program are to develop pharmacological probes of methyl-lysine binding domains to pioneer both the validation of specific domains and the assay framework in which issues of selectivity and mechanism of action can be assessed. We have discovered small molecule ligands for the methyl-lysine readers, L3MBTL3 and L3MBTL1, with low micro-molar to nanomolar Kd values (ITC). Ligands for L3MBTL3 also demonstrate effects on L3MBTL3- GFP protein localization in whole cells. We propose the optimization of these series to provide high quality chemical probes. The malignant brain tumor (MBT) repeat is a structural domain of ca. 100 amino acids and occurs in 9 human proteins that recognize mono- and dimethyl-lysine modifications of histones. There are no high-quality chemical probes for MBT domains, or indeed, any other methyl-lysine binding domain. Current understanding of the biological consequences of MBT domain antagonism would suggest that antagonists may be useful in de-differentiation, re-expression of silenced genes and cellular reprogramming, We will establish a firm connection between methyl-lysine reader in vitro antagonism, cell-based localization of the targeted reader, genome-wide selectivity finger-printing and biological consequences of reader antagonism. The probes developed in the course of this research would be made freely available to the academic community with no restrictions on use or intellectual property constraints.